The dopamine system is an extremely important system for essential regulation of locomotion, hormone secretion, emotions and such in the mammalian brain. Thus, abnormalities in dopaminergic neural transmission cause various neural disorders. For example, Parkinson's disease (PD) is a neurodegenerative disease of the extrapyramidal system that occurs due to specific degeneration of dopaminergic neurons in the substantia nigra of the midbrain (Harrison's Principles of Internal Medicine, Vol. 2, 23rd edition, Isselbacher et al., ed., McGraw-Hill Inc., NY (1994), pp. 2275-7). Oral administration of L-DOPA (3,4-dihydroxyphenylalanine) is performed as a primary therapeutic method for Parkinson's disease to compensate for the decrease in the amount of dopamine produced; however, the duration of the effect is known to be unsatisfactory.
Therapeutic methods have been attempted whereby the midbrain ventral regions of 6- to 9-week old aborted fetuses, which contain dopaminergic neuron progenitor cells, are transplanted to compensate for the loss of dopaminergic neurons (U.S. Pat. No. 5,690,927; Spencer et al. (1992) N. Engl. J. Med. 327: 1541-8; Freed et al. (1992) N. Engl. J. Med. 327: 1549-55; Widner et al. (1992) N. Engl. J. Med. 327: 1556-63; Kordower et al. (1995) N. Engl. J. Med. 332: 1118-24; Defer et al. (1996) Brain 119: 41-50; Lopez-Lozano et al. (1997) Transp. Proc. 29: 977-80). However, these methods currently present problems relating to cell supply and ethics (Rosenstain (1995) Exp. Neurol. 33: 106; Turner et al. (1993) Neurosurg. 33: 1031-7). Also, various problems are being pointed out, such as the risk of infection and contamination, immunological rejection of transplants (Lopez-Lozano et al. (1997) Transp. Proc. 29: 977-980; Widner and Brudin (1988) Brain Res. Rev. 13: 287-324), and low survival rates due to the primary dependence of fetal tissues on lipid metabolism rather than glycolysis (Rosenstein (1995) Exp. Neurol. 33: 106).
In order to resolve ethical issues and the shortage of supply, methods have been proposed that use, for example, porcine cortex, stria, and midbrain cells (for example, Japanese Patent Kohyo Publication No. (JP-A) H10-508487, JP-A H10-508488 or JP-A H10-509034). In these methods, a complex procedure involving the alteration of cell surface antigens (MHC class I antigens) is required to suppress rejection. Methods involving local immunosuppression by the simultaneous transplantation of Sertoli's cells have been proposed as a method for eliminating transplant rejection (JP-A H11-509170, JP-A HI11-501818, Selawry and Cameron (1993) Cell Transplant 2: 123-9). It is possible to obtain transplant cells from relatives with matching MHCs, bone marrow from other individuals, bone marrow banks, or umbilical cord-blood banks. However, if it were possible to use the patient's own cells, the problem of rejection reactions could be overcome without any laborious procedures or trouble.
Therefore, instead of using cells from aborted fetuses as transplant materials, the use of dopaminergic neurons differentiated in vitro from non-neural cells such as embryonic stem (ES) cells and bone marrow interstitial cells is considered to be promising. In fact, functional dopaminergic neurons were reported to have been formed upon transplanting ES cells to lesion stria of a rat Parkinson's disease model (Kim et al. (2002) Nature 418: 50-56). It is believed that regenerative therapy from ES cells or a patient's own nerve stem cells will be increasingly important in the future.
In the treatment of nerve tissue damage, brain function must be reconstructed, and in order to form suitable links with surrounding cells (network formation), it is necessary to transplant immature cells, which can differentiate in vivo into neurons. When transplanting neuron progenitor cells, in addition to the aforementioned problem regarding supply, there is also the possibility that the progenitor cells will differentiate into groups of heterogeneous cells. For example, in treating Parkinson's disease, it is necessary to selectively transplant catecholamine-containing neurons that produce dopamine. Examples of transplant cells proposed in the past for use in the treatment of Parkinson's disease include striatum (Lindvall et al. (1989) Arch. Neurol. 46: 615-31; Widner et al. (1992) N. Engl. J. Med. 327: 1556-63), immortalized cell lines derived from human fetal neurons (JP-A H08-509215; JP-A H11-506930; JP-A No. 2002-522070), human postmitotic neurons derived from NT2Z cells (JP-A H09-5050554), primordial neuron cells (JP-A H11-509729), cells and bone marrow stroma cells transfected with exogenous genes so as to produce catecholamines such as dopamines (JP-A 2002-504503; JP-A 2002-513545), and genetically engineered ES cells (Kim et al. (2002) Nature 418: 50-56). However, none of these contain only dopaminergic neurons or cells that differentiate into dopaminergic cells.
A method has been proposed for selectively concentrating and isolating dopaminergic neurons from undifferentiated cell populations. In this method, a reporter gene expressing a fluorescent protein is introduced into each cell of a cell population, under the control of a gene promoter/enhancer, such as the tyrosine hydroxylase (TH) expressed in dopaminergic neurons. Fluorescing cells are then isolated. The dopaminergic neurons are visualized in their viable state, and concentrated, isolated, and identified (Japanese Patent Application Kokai Publication No. (JP-A) 2002-51775 (unexamined, published Japanese patent application)). This method requires the complicated step of introducing an exogenous gene. Further, the presence of a reporter gene poses problems of toxicity and immunogenicity when used in conjunction with gene therapy.
Lmx1a was identified as a LIM-type homeobox gene expressed in the roof plate of the developing spinal cord (the organizer region which secretes differentiation-inducing factors in the most dorsal region; neurons do not develop from this region), the neural crest, the hindbrain rhombic lip, and the posterior region of the developing cerebral hemisphere (Non-Patent Documents 1 and 2). In dreher mutant mice, which are animal models for type II agyria, autosomal recessive mutations have been revealed to occur on Lmx1a (Non-Patent Document 1). Furthermore, it is also known that mutations in Lmx1a trigger lesions in the central nervous system in queue courte (qc) rats (Non-Patent Documents 3 to 5). The Lmx1a gene is expressed not only during the fetal phase but also after birth, and contributes to the formation of the cerebral cortex and the cerebellum.    Non-Patent Document 1: Millonig et al. (2000) Nature 403: 764-769    Non-Patent Document 2: Failli et al. (2002) Mechanisms of Development 118(1-2): 225-228    Non-Patent Document 3: Kitada et al. (2001) 2001 Meeting on Physiological Genomics and Rat Models    Non-Patent Document 4: Kitada et al. (2001) The 15th International Mouse Genome Conference    Non-Patent Document 5: Kitada et al. (2000) Record of the 17th Meeting of the Japanese Society of Animal Models for Human Diseases